Apoptosis and autophagy are both tightly regulated cellular processes that play crucial roles in life and death decisions of the cell, tissue homeostasis, development, and cancer biology. The first discovered anti-apoptotic protein was Bcl-2, and our laboratory identified the first mammalian autophagy protein, Beclin 1. In the previous project period, we demonstrated that Bcl-2 functions as an anti-autophagy protein, and identified a key biochemical mechanism (i.e. stress-induced Bcl-2 multisite phosphorylation) underlying the regulation of interactions between Bcl-2 and Beclin 1. During the next project period, we propose to gain a deeper understanding of both the molecular mechanisms regulating the Bcl-2/Beclin 1 interaction, and the in vivo physiological significance of Bcl-2 regulation of Beclin 1-dependent autophagy. In the first specific aim, we will use structural biology, biochemical, and cell biology approaches to define novel molecular determinants that regulate the interaction between Bcl-2 and Beclin 1. We will use nuclear magnetic resonance (NMR) to identify residues in cellular Bcl-2 that are predicted to be selectively involved in binding to Beclin 1 (but not other BH3 domain containing proteins), test these predictions in functional assays, and perform biochemical and cell biology studies to investigate whether starvation-regulated post-translational modifications of Beclin 1 that we have identified in preliminary studies (e.g. serine phosphorylation, arginine methylation) are: (1) regulated by Bcl-2; (2) modify Bcl-2/Beclin 1 interactions; and (3) modify Beclin 1 autophagic activity. In the second specific aim, we will evaluate the in vivo physiological significance of Bcl-2 modulation of the autophagy function of Beclin 1, using targeted mutant mice that express mutant forms of either Bcl-2 or Beclin 1 that alter the normal regulation of the Bcl-2/Beclin 1 interaction. We will characterize previously generated Bcl-2 nonphosphorylatable mutant mice that are predicted to constitutively inhibit Beclin 1 function (as well as newly generated mice based on our results with studies in the first specific aim) with respect to in vivo regulation of autophagy and autophagy-dependent biological processes. Together, these studies are expected to help elucidate the molecular mechanisms underlying the control of autophagy by the Bcl-2/Beclin 1 complex and the significance of this complex in regulating life and death decisions of the cell, tissue homeostasis, development, and cancer biology.